The human body is composed of about 200 kinds of 100 trillion cells, in which the physiological activity is regulated by the action of various proteins to maintain life.
Cells are surrounded by membranes in bilayer structure composed of phospholipids, which block the entry of foreign substances into cells. Most of the protein drugs which have developed so far cannot pass through the cell membrane to enter the cell and can act on the outside of the cell or act on a receptor on the cell membrane to deliver the signal into the cell in order to show physiological effect.
Cytosol has lots of proteins which interact with each other to regulate physiological activity. So, if only a protein drug can be delivered inside the cell, that is, inside the cytosol, the cell activity would be controlled more effectively.
Recently, studies have been actively going on to establish a method for delivering a cargo protein directly into cells via cell membrane. When a recombinant protein of a cargo protein and protein transduction domains (PTDs), the peptide that passes through the cell membrane, is prepared and administered, it can enter the cytosol through the cell membrane (FIG. 1). PTD is exemplified by HIV-1 TAT, HSV VP22, Antp, dfTAT, and Hph-1. A fusion protein prepared by combining the PTDs and a cargo protein is produced as a recombinant protein and at this time a separation process is required. However, this process is problematic in that the protein refolding is not performed properly, the activity is decreased, the protein is nonspecifically transferred, the risk of causing an immune reaction in vivo is large, the cost is high, and the yield is low.
The cargo protein conjugated with various nanoparticles can enter the cytosol through the cell membrane by endocytosis (FIG. 2). At this time, the nanoparticles are exemplified by Gold NP, Liposome NP, Magnetic NP, and Polymeric NP, etc. The separation of the nanoparticles from the cargo protein occurs mostly in lysosome in the cell, so the cargo protein is decomposed inside lysosome to lose its activity. Or the nanoparticles are difficult to be separated from the cargo protein in cytosol and toxicity of the nanoparticles can be another problem.
Exosome is a small vesicle with a membrane structure in the size of 50˜200 nm, which is secreted out of the cell with containing protein, DNA, and RNA for intercellular signaling.
Exosome was first found in the process of leaving only hemoglobin in the red blood cells by eliminating intracellular proteins at the last stage of red blood cell maturation. From the observation under electron microscope, it was confirmed that exosome is not separated directly from plasma membrane but discharged extracellular from the intracellular specific zone, called multi-vesicular bodies (MVBs). That is, when MVBs are fused with plasma membrane, such vesicles are discharged outside of the cell, which are called exosome (FIG. 3).
It has not been clearly disclosed the molecular mechanism of the exosome generation. However, it is known that various immune cells including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, and macrophages, stem cells, and tumor cells produce and secrete exosomes when they are alive.
Exosome contains various intracellular proteins, DNA, and RNA. The substances secreted out of the cells contained in these exosomes can be reintroduced into other cells by fusion or endocytosis and serve as intercellular messengers. By analyzing such substances that are secreted out of the cell as included in exosome, specific disease can be diagnosed.
Exosome also includes various types of microRNAs. A method for diagnosing a disease by detecting the presence or absence and the abundance thereof has been reported (KR 10-2010-0127768A). International Patent Publication No. WO2009-015357A describes a method for predicting and diagnosing a specific disease by detecting exosome in the cancer patient originated samples (blood, saliva, tears, etc.). In particular, the exosome obtained from a patient having a specific disease (lung disease) is analyzed and the relationship between a specific microRNA and lung disease is specifically described. Studies have been still going on to establish a method to diagnose kidney disease, in addition to lung disease, by using a specific protein included in exosome.
Exosome might also include antigens. In antigen presenting cells (APC), antigen peptide is loaded in MHC (major histocompatibility complex) class II molecule in the intracellular compartment having a membrane structure including polycystic bodies. Therefore, the exosome originated therefrom also has the antigen peptide-MHC class II complex. So, exosome acts as an immunogen carrier to present antigen peptide to CD4+T lymphocytes and thereby can induce immune response such as T lymphocyte proliferation. The molecules that are able to stimulate immune response such as MHC class I and heat-shock proteins (HSPs) are concentrated in exosome, so that exosome can be used to increase or decrease immune response for the treatment of cancer or auto-immune disease.